Apparatus and method for ultrasonic treatment of a liquid

ABSTRACT

The present invention is an apparatus and method for ultrasonically treating a liquid to generate a product. The apparatus is capable of treating a continuously-flowing, or intermittently-flowing, liquid along a line segment coincident with the flow path of the liquid. The apparatus has one or more ultrasonic transducers positioned asymmetrically about the line segment. The ultrasonic field encompasses the line segment and the ultrasonic energy may be concentrated along the line segment. Lysing treatments have been successfully achieved with efficiencies of greater than 99% using ultrasound at MHz frequencies without erosion or heating problems and without the need for chemical or mechanical pretreatment, or contrast agents. The present invention overcomes drawbacks of current ultrasonic treatments beyond lysing and opens up new sonochemical and sonophysical processing opportunities.

This invention was made with Government support under ContractDE-AC0676RLO1830 awarded by the U.S. Department of Energy. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention is an apparatus and method for ultrasonicallytreating a liquid-based medium to generate a product. The liquid-basedmedium, hereinafter referred to simply as “liquid,” means single-phaseliquids having one or more constituents, as well as liquid-solidmixtures such as suspensions, dispersions, slurries, colloids, andbiological tissue.

BACKGROUND OF THE INVENTION

Ultrasound is a form of vibrational energy. When it propagates through,and interacts with, a liquid, the energy is attenuated by scattering orabsorption. At low ultrasound powers, the energy is absorbed by theliquid in a thermal interaction that causes local heating. At higherpowers the interaction becomes increasingly non-linear and bothnon-thermal mechanical and cavitational mechanisms become significant.The non-thermal mechanical mechanisms can include radiation pressure,acoustic streaming, radiation forces, torques, and near-boundary/bubblehydrodynamic shear forces.

These ultrasonic interactions with a liquid, particularly thoseinvolving cavitation, have been exploited for many years in devices thatclean or separate materials, accelerate or modify chemical reactions,and kill or lyse cells. Such devices typically utilize sonic horns, orprobes, and are designed to optimize the cavitation mechanism atfrequencies generally in the range of 20-50 kHz. For comparison,ultrasound devices used in the medical field typically operate atfrequencies of 0.8-15 MHz and at lower power densities (<0.5 W/cm² fordiagnostics and ˜0.5-3 W/cm² for therapy).

Ultrasound offers an attractive cell lysing tool to obtain sufficientamounts of nuclear, cytoplasmic, or other cellular material forcommercial use (e.g., proteins), or for analysis and identification(e.g., anthrax or e-coli). Effective and rapid lysing is particularlyimportant for the most refractory microorganisms of concern to publichealth including protozoan cysts, fungal hyphae, Gram positive bacteria,and spores. In a suspension containing microorganisms, the nature of theultrasound-suspension interaction is complex and has been shown todepend on at least the power level in the ultrasound, the ultrasoundfield geometry, and frequency of the ultrasound.

Current ultrasound lysing (and other material processing) devicestypically use kHz frequencies with a horn or probe configured tooptimize cavitation. For a given frequency, there is a minimum powerlevel necessary to cause cavitation, known as the cavitation threshold.In general, the power necessary to achieve cavitation increases withfrequency. Thus, when using MHz frequencies, contrast agents (e.g.,microbubbles, microparticles) are often introduced in the liquid to helpreduce the cavitation threshold by increasing the mechanical interactionand inducing cavitation-like phenomena. In some MHz applications, it isonly with the presence of such contrast agents that cavitation occurs.

Because ultrasonic vibration is rapidly attenuated in passing throughlong paths in a liquid, it is common to effect cell lysis by applyingthe cavitating kHz ultrasound in a confined chamber. Current soniclysing devices typically employ a batch processing approach using staticliquid reaction chambers. For example, Belgrader et al (Anal. Chemistry,Vol. 71, No. 19, Oct. 1, 1999) employs a horn-based minisonicator forspore lysis and subsequent polymerase chain reaction analysis. Suchdevices are prone to erosion of the sonic horn tip and unacceptableheating of the liquid.

A few flow-through devices have been developed, though they stillincorporate sonic probes depositing energy in a confined chamber. Forexample, the flow-through devices disclosed in U.S. Pat. No. 3,715,104and McIntosh and Hobbs (Proc. of Ultrasounics in Industry, pp 6-8, Oct.20-21, 1970) agitate a liquid between two closely spaced flat surfaces.Furthermore, T. J. Mason (Ultrasonics, 1992, Vol. 30, No. 3, pp 192-196)discloses other flow-through sonic devices that incorporate transducerssymmetrically positioned about the flow path of a liquid.

Most current ultrasound processing devices, however, cannot meet thepractical, economical, and operational requirements associated withindustrial-scale chemical/physical processing systems, field deployablesystems, or continuous biomonitoring systems. This is especially truefor systems requiring automation or remote operation. Such systemsrequire rapid, effective, efficient, and near-continuous processing withminimal or no manual steps. As the present invention will illustrate,there is an opportunity to apply non-conventional combinations ofultrasonic power, frequency, and field geometry to address currentlysing needs and to improve existing (and develop new) chemical andphysical processing methods for materials. In particular, ultrasonictreatment at conditions that avoid conventional cavitation and promotenon-thermal mechanical interactions shows great potential.

BRIEF SUMMARY OF THE INVENTION

The present invention is an apparatus and method for ultrasonicallytreating a liquid to generate a product. The apparatus is capable oftreating a continuously-flowing, or intermittently-flowing, liquid alonga line segment coincident with the flow path of the liquid. Theapparatus has one or more ultrasonic transducers positionedasymmetrically about the line segment. The term ‘asymmetric’ as usedherein in relation to asymmetric positioning of transducers meansradially asymmetric orthogonal to the axis of the line segment. Theultrasound field encompasses the line segment and the ultrasonic energymay be concentrated along the line segment Lysing treatments have beensuccessfully achieved with efficiencies of greater than 99% usingultrasound at MHz frequencies without the typical cavitation andassociated problems, and without the need for chemical or mechanicalpretreatment, or contrast agents.

An object of the present invention is to ultrasonically treat acontinuously-flowing or an intermittently-flowing liquid to generate aproduct.

A further object of the present invention is to maximize the amount ofproduct generated for a given transducer power input.

A further object of the present invention is to provide a rapid,effective, and field-deployable ultrasonic treatment system thatrequires minimal manual intervention.

A further object of the present invention is to lyse cells, producingavailable nuclear, cytoplasmic, or other cellular material, with greaterthan 80% efficiency.

A further object of the present invention is to lyse cells in a liquidthat does not require chemical or physical pretreatment, or contrastagents.

A further object of the present invention is to improve the productivityof sonochemical and sonophysical treatments that have traditionally beenbased on batch processing.

The subject matter of the present invention is particularly pointed outand distinctly claimed in the concluding portion of this specification.However, both the organization and method of operation, together withfurther advantages and objects thereof, may best be understood byreference to the following description taken in connection withaccompanying drawings wherein like reference characters refer to likeelements.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an illustration of the present invention with a cylindricalsonic coupler;

FIG. 1B is an illustration of the present invention with a triangularsonic coupler;

FIG. 1C is an illustration of the present invention with a rectangularsonic coupler;

FIG. 2 is an illustration of the present invention with a reaction tube;

FIG. 3 is an illustration of a static liquid chamber used in lysisexperiment 1;

FIG. 4 is an illustration of a flow through chamber used in lysisexperiment 2;

FIG. 5A is a side view of the full cylinder transducer configurationused in experiment 3;

FIG. 5B is an end view of the full cylinder transducer configurationused in experiment 3;

FIG. 6A is a side view of the piezoelectric element;

FIG. 6B is an end view of the piezoelectric element;

FIG. 7A is a side view of the flow-through device with sonic energyconcentrated along a line segment used in lysis experiments 4 and 5; and

FIG. 7B is an end view of the flow-through device with sonic energyconcentrated along a line segment used in lysis experiments 4 and 5.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is an apparatus and method for ultrasonicallytreating a liquid to generate a product. The liquid may be asingle-phase liquid having one or more constituents (e.g.,chemical/petrochemical solutions and biological liquids such as bloodplasma and urine) as well as liquid-solid mixtures such as suspensions,dispersions, slurries, colloids, and biological tissue. The liquid-solidmixture may comprise biological material selected from the groupconsisting of microorganisms, cells, viruses, tissues, and combinationsthereof. The product includes, but is not limited to, available nuclear,cytoplasmic, and other cellular material from lysed cells and othermaterials used in industry that are activated, crystallized,precipitated, sterilized, extracted, impregnated, dispersed, defoamed,degassed, deaggregated, homogenized, or emulsified by the ultrasonicinteraction.

The apparatus is capable of treating a continuously-flowing orintermittently-flowing liquid. For example, the process may require acontinuously-flowing liquid to optimize the reaction that generates theproduct, maintain a high operational productivity, or to maintainvigilance in monitoring a biological or chemical threat. Intermittentflows may be implemented in those circumstances where continuous batchtreatments are desired.

Several embodiments of the present invention are shown in FIGS. 1A-1C.The liquid to be treated flows along the flow path 108 through thedevice 100. The liquid in the device 100 is exposed to a uniqueultrasound field produced by one or more ultrasound-producingtransducer(s) 104 positioned asymmetrically about the flow path 108. Theultrasonic field encompasses the flow path 108 of the liquid within thedevice 100 including a line segment 106 (see FIG. 1A, not shown in FIGS.1B-1C for clarity) that is coincident with the longitudinal axis of theflow path 108. The transducer(s) 104 is acoustically coupled to theliquid by a sonic coupler 110. In the embodiments of FIGS. 1A-1B, thetransducer(s) are positioned so as to concentrate sonic energy along theline segment 106. The transducer(s) 104 include piezoelectric,magnetorestrictive, and other devices capable of producing an ultrasonicfield. The line segment 106 may be straight or curved. For example, theline segment 106 (and flow path 108) may be helical to increase theresidence time (and thus, treatment time) of the liquid in the sonicfield.

In these embodiments, the sonic coupler 110 is a solid material that maybe rigid or flexible, and provides the flow path 108 for the liquid (theliquid may enter and exit the device 100 along the flow path 108 byconnecting tubing or piping (not shown) to the entrance and exit of thedevice 100). It is preferable that the sonic coupler 110 is made of amaterial with a low attenuation coefficient to avoid overheating of thesonic coupler 110 and has an acoustical impedance value between theacoustical impedance of the liquid and that of the transducer(s) 104.For example, aqueous liquids have an acoustical impedance ofapproximately 1.5×10⁶ kg/m²/s and piezoelectric transducer materials(e.g., high density ceramics) typically have acoustical impedances inthe range of 20×10⁶−36×10⁶ kg/m²/s. Thus, candidate sonic coupler 110materials include metals (e.g., aluminum), ceramics, glasses, minerals,and combinations thereof. Due to the various geometries that may berequired to obtain an asymmetric positioning of the transducer(s) 104,it is preferable that the sonic coupler 110 is easily machinable such asa machinable ceramic. Machinable ceramics include glass-mica (e.g.,MACOR®, MACOR is a registered trademark of Corning Glass Works),boron-nitrate, aluminum silicate, alumina bisque, and combinationsthereof.

It is more preferable that the sonic coupler 110 is made of a materialwith an acoustical impedance value approximately equal to the geometricmean of the acoustical impedances of the liquid and the transducer(s)104. For example, if the liquid and transducer(s) 104 have acousticalimpedances of 1.5×10⁶ kg/m²/s and 30×10⁶ kg/m²/s, respectively, amaterial having an acoustical impedance of (1.5×30)^(1/2)=6.7 would bemore preferred.

As known to those skilled in the art, there are various methods toensure an adequate acoustical coupling between the transducer(s) 104 andthe sonic coupler 110 itself. For example, the two components may beepoxied together or machined to a close fit and smooth surface finishthat minimizes loss of ultrasound energy associated with gaps betweenthe two components.

Though the embodiments of the present invention shown in FIGS. 1A-1Cillustrate cylindrical, triangular, and rectangular sonic couplers 110and various numbers of cylindrical and planar transducer(s) 104, thepresent invention is not limited to such shapes and numbers. Forexample, the sonic coupler 110 may be hexagonal (or oval) with one ormore planar transducer(s) 104 asymmetrically positioned on the soniccoupler 110. In addition, the transducer(s) 104 may comprise a singletransducer and be concave with the sonic coupler 110 machined or shapedto accommodate such transducer(s) 104 geometry.

An alternative embodiment of the present invention is the device 100shown in FIG. 2. In this embodiment, the sonic coupler 110 of FIGS.1A-1C is replaced by a reaction tube 202 and a reaction tube coupler204. The reaction tube 202 provides the flow path 108 for the liquid andis acoustically coupled to both the liquid and the reaction tube coupler204. As in the previous embodiments, the reaction tube coupler 204 isacoustically coupled to the transducer(s) 104. The reaction tube 202 maybe made of any structural material that is compatible (e.g., chemically)with the fluid and reaction tube coupler 204 including, but not limitedto, metal, glass, and plastic. In this embodiment, the reaction tubecoupler 204 can be made of the same material (and in the same shape) asthe sonic coupler 110 of the embodiments shown in FIGS. 1A-1C or it canbe a liquid, preferably water. Though the reaction tube coupler 204 isillustrated as being cylindrical in FIG. 2, the present invention is notlimited to such a shape, especially if the reaction tube coupler 204 isa liquid. In such circumstances, a requirement is that the liquidprovide sufficient acoustical coupling between the reaction tube 202 andthe reaction tube coupler 204 (for example, by immersing the reactiontube 202 and the reaction tube coupler 204 in a liquid bath).

Furthermore, though the embodiments of the present invention shown inFIGS. 1A-1C and FIG. 2 illustrate a single flow path 108, the presentinvention is not limited to a single flow path 108. That is, it isapparent that multiple flow paths could be incorporated in the device100 of FIGS. 1A-1C and FIG. 2 (e.g., to increase the volumetricprocessing or treatment rate of the liquid).

The following successful experiments, with the notable exception ofexperiment 3, illustrate new combinations of ultrasound power,frequency, and field geometry that meet the challenge of lysing Bacillusglobigii (BG) spore suspensions. Such successful lysis experiments arenot intended to limit the present invention to such a specificbiological treatment. It will be apparent to those skilled in the artthat the present invention overcomes drawbacks of current ultrasonictreatments beyond lysing and opens up new sonochemical and sonophysicaltreatments for material processing industries including, but not limitedto, chemical, biochemical, petrochemical, food, and mining.

Furthermore, though some details on how the fluid in the presentinvention is made to flow through the device 100 are provided below,such features should not be interpreted as limitations to the presentinvention. That is, there are many different mechanisms to cause theliquid to flow (pumps, pneumatic, gravity feed, etc.) and a variety ofpiping and valving arrangements to have the liquid flow intermittently.The variety of components and arrangements to accomplish fluid flow inthe present invention are known to those skilled in the art of fluidsystems.

Experimental Procedure

BG spore suspensions originated from stock preparations maintained atDugway Proving Grounds. Spores were resuspended in sterile water andsubjected to several rounds of vigorous mixing, settling and decantingto eliminate spore clumps. Plate counts and microscopy were used toconfirm the consistency of the stock spore suspension and verify that asingle spore gave rise to a single colony. A suspension of 10⁸ spores/mlwas used for all lysis experiments described below.

Ten-fold serial dilutions of spore suspension were prepared in sterilewater within two hours of all lysis experiments. Three×20 μl aliquotswere spotted directly onto trypticase soy agar (Difco, Detroit, Mich.)plates and incubated at 30° C. for 36 hours. All lysis and platingexperiments were performed in triplicate, resulting in at least 9 datapoints (spore counts) for each treatment. Lysis efficiency wascalculated as 100×(C_(o)−C_(lysis))/C_(o); where C_(o) is the viablespore count before lysis and C_(lysis) is the viable spore count afterlysis.

The original spore preparation had significant quantities of adsorbeddeoxyribonucleic acid (DNA) on the spore coat that interfered withpolymerase chain reaction (PCR) detection of intracellular or liberatedBG DNA. Eight hundred microliter aliquots of BG spores were collected bycentrifugation and resuspended in 200 μl 10% sodium hypochlorite for1-10 minutes. After this decontamination, spores were recovered bycentrifugation, washed extensively in sterile water, and then subjectedto on-line lysis (described in the experiments below) and PCRamplification. Control lysis experiments showed that the hypochloritetreatment had no effect on cell lysis efficiency, spore viability, orcarryover of PCR inhibitors.

DNA availability after spore lysis was assessed by adilution-to-extinction PCR method. Genomic DNA was isolated from BGspores by bead-mill homogenization and quantified by ultravioletspectrophotometry. PCR primers Bg215f and Bg325r were provided by NavyMedical Research Institute and synthesized by Keystone Labs (Camarillo,Calif.). PCR amplification was carried out in 25 μl total volume,utilizing an M J Research (Watertown, Mass.) Tetrad thermal cycler and0.2 ml thin-walled reaction tubes. Spore preparations (lysed or unlysed)were serial diluted in a 10-fold series immediately prior to PCR, andpurified BG DNA was serially diluted in PCR-grade water at 100 pg to 1fg μl-1 as a positive control template. Final reaction conditions were 5μl DNA or lysed/unlysed spore preparation, 10 mM Tris pH 8.3, 50 mM KCl,2.5 mM MgCl2, 200 μM each dNTP, 0.2 μM each primer, 5 μl template DNA orlysed spore suspension, and 0.625 U LD-Taq polymerase (Perkin Elmer,Foster City, Calif.) which had been pretreated with TaqStart™ antibodyat the recommended concentration (Sigma, St. Louis, Mo.). Assembledreactions were amplified with 45 cycles at 94° C. for 15 s, 56° C. for30 s, 72° C. for 30 sec with a 2 s extension per cycle. The entirecontents of each PCR were analyzed on 1% NuSieve, 1% Seakem GTG agarose(FMC Bioproducts, Rockland, Me.) gels in 1×TAE running buffer, bothcontaining ethidium bromide, and gel images captured with a BioRad(Hercules, Calif.) Fluor-S imager and Molecular Analyst software.

Experiment 1

Experiment 1 demonstrated that cell lysis can be performed with greaterthan 99% efficiency using 1 MHz ultrasound both with and without theaddition of contrast agents. FIG. 3 shows an initial static-liquidchamber lysis experiment using 1 MHz ultrasound, whereby the bottom of astandard polypropylene microfuge tube 300, containing a 200 microlitersuspension of BG, was held in place in a water bath 308 in the sonicfield 304 produced by the transducer 312 in an ultrasonic humidifier316. The angle of the microfuge tube, θ, was set to zero (i.e., the tubewas vertical) for this particular experiment. The specific humidifier316 was a Holmes Ultrasonic humidifier, model HM-460B, ca. 10 W/cm² peakpower. Sixty milligrams of 50 micron glass microspheres and 40micrograms of paramagnetic particles were added (in separatesubexperiments) to help induce cavitation and/or enhance collisionrates.

Though this experiment did not utilize a flowing liquid, the results,shown in Table 1 below, clearly indicate that cell lysis can be obtainedin a MHz sonic field. The presence or absence of microparticles had noappreciable effect on spore lysis efficiency or in-tube temperature,suggesting that the added microparticles were neither acting as anenergy sink nor microbubble source during the course of the experiment.This system also caused up to a 5-log reduction in cell viability forvegetative Escherichia coli, Bacillus aetrophaeus and Bacillusthuringiensis kurstaki cells, with extensive cellular damage asdetermined by light microscopy. All subsequent experiments weretherefore performed without microparticles, as would be advantageous inan automated biomonitoring system.

TABLE 1 Percent lysis efficiency of BG spores in a static liquidvertical chamber using MHz ultrasound. 4 × 30 sec with: % LysisEfficiency In-Tube Average Temp. (° C.) No Beads 99.4 104 Glass Beads99.6 104 Magnetic Beads 99.1 103

Experiment 2

The first “flow-through” sonic device experiment is illustrated in FIG.4, whereby the same humidifier 316, transducer 312, and water bath 308as that of experiment 1 was used. In experiment 2, however, a reactiontube 202 made of 3.2 mm OD×1.5 mm ID TEFLON® (i.e.,polytetrafluoroethylene, TEFLON is a registered trademark of E. I.Dupont de Nemours and Company) and one made of 3.2 mm OD×1.5 mm IDpolyetherethylketone (PEEK) chromatography tubing were used in twoseparate subexperiments. Note that in this experiment, the water bath308 functions as the reaction tube coupler 204 of FIG. 2. The reactiontube 202 was connected to a standard sequential injection system (FiaLab3000, Alitea, USA) that included a 1 ml syringe pump (Cavro, Sunnyvale,Calif.) and a 10-port selection valve (Valco, Cheminert, Houston, Tex.).The flow injection system delivered a continuous flow (at 1 μl/s and 5μl/s) of BG spore suspension through the reaction tube 202 that waspartially immersed in the 1 MHz sonic field 304 produced by thetransducer 312. As shown in FIG. 4, the sonic field 304 encompassed aline segment 106 (not shown for clarity) coincident with the flow path108 in the trough region of the reaction tube 202. The line segment 106was approximately 5 mm in length, resulting in 0.5 to 2.5 secondexposure of spores to the sonic field as opposed to the 1 to 2 minutesof exposure employed in the previous batch experiment 1. Also introducedwere 15 μl air segments every 10 μl of spore suspension to mimic theair/liquid interface present in experiment 1. In all cases, the totalvolume of spore suspension processed was 200 μl.

Results from this experiment are shown in Table 3 below, whichdemonstrate that a MHz sonic field can effect spore lysis in anear-instantaneous manner. Air segmentation appeared to significantlyenhance lysis efficiency in the TEFLON tubing, but this interaction wasnot pursued because TEFLON tubing melted during the course of the trials(40-200 seconds continuous power). PEEK tubing, on the other hand,withstood the sonic energy and maintained in-tube temperatures near 100°C., but air segmentation had little effect on lysis efficiency. Controlplating experiments (no lysis) showed that BG spores were not retainedwithin the fluidics system, such that all plate counts associated withTable 3 reflect treatment effects on BG spores rather thancross-contamination and carryover between lysis trials. All furtherexperiments were conducted in PEEK tubing in the absence of any airsegmentation or microbubble amendments, again compatible with a fielddeployable and automated biomonitoring system.

TABLE 3 Percent lysis efficiency of BG spores in a flow-through MHzlysis experiment. No Air Plus Air TEFLON TUBING 1 μl sec⁻¹ 82.4 99.3 5μl sec⁻¹ 21.2 72.0 PEEK TUBING 1 μl sec⁻¹ 90.4 88.9

Experiment 3

The previous two experiments show that the liquid residence time in thesonic field is an important variable for effective cell lysis.Consequently, a device wherein the sonic energy is concentrated along aline segment 106 throughout the entire length of the transducer(s) 104was developed as shown in FIGS. 5A-5B.

The transducer(s) 104 was a commercial 1.48 MHz cylindricalpiezoelectric element shown in FIGS. 6A-6B and placed in high densityfoam 600 to absorb the energy radiating outward from the diameter of thetransducer(s) 104. A Hewlett Packard 33120A 15 MHz function/arbitrarywaveform generator was used and frequency was monitored with an ENIA-300 RF power amplifier and Tektronix TDS 460-A 4 channel digitizingoscilloscope. It was presumed that such a symmetrical configurationwould provide a high-intensity field along the line segment 106 (i.e.,along the longitudinal axis of the transducer(s) 104) for subsequentprocessing of a fluid flowing along the line segment 106.

The entire configuration was immersed in water. When power was appliedto the transducer(s) 104, only a small amount of acoustic activity wasobserved near the ends of the transducer(s) 104. While ultrasonicpressure may have been developed along the line segment 106, no acousticstreaming was observed and it did not appear to provide sufficient sonicenergy desired for lysis. This is understandable based on the effect ofacoustic wave cancellation caused by the symmetry of the device.

Experiment 4

Whereas the previous experiment demonstrated unsatisfactory sonic energywith a symmetrical configuration, an asymmetric configuration (shown inFIGS. 7A-7B) was demonstrated to provide sufficient energy. In thisembodiment, the device 100 used a transducer(s) 104 in the form of apartial cylinder representing less than half the full cylinder used inexperiment 3. Based on the results of experiment 3, if thecross-sectional arc α is greater than 180°, the sonic field generatedwould oppose itself at the opposite side of the cylinder and decreasethe field intensity along the longitudinal axis of the transducer(s)104. Therefore, the transducer(s) 104 was fabricated from a section ofthe commercial 1.48 MHz cylindrical piezoelectric element (FIGS. 6A-6B)with a cross-sectional arc, α, equal to 160° (FIGS. 7A-7B). Thetransducer(s) 104 was placed in high density foam 600 to absorb theenergy radiating outward from the diameter of the transducer(s) 104.This asymmetric geometry was used to concentrate sonic energy along theline segment 106 coinciding with the longitudinal axis of the reactiontube 202. The reaction tube 202, made of 3.2 mm OD×1.5 mm ID PEEKtubing, was positioned approximately at the central axis of thetransducer(s) 104. In such a configuration, liquid residence time andtemperatures in the reaction tube 202 are a function of flow rate, andincident sonic energy a function of power, frequency, and distance fromthe transducer(s) 104. Temperatures in the reaction tube 202 wererecorded with a thermocouple (not shown). Acoustic power intensity wasmeasured at various points in the sonic field with a calibrated pintransducer (not shown).

The entire configuration was immersed in water (serving as the reactiontube coupler 204, not shown) to acoustically couple the transducer(s)104 to the reaction tube 202. Under the proper driving power, thisconfiguration gave excellent lysis results. Two hundred microliteraliquots of spore suspension were flowed through the reaction tube 202at 1 μl sec⁻¹ at variable power (700, 800, 900 and 1000 mV) and atvariable distances between the reaction tube 202 and transducer(s) 104.Lysis efficiencies of greater than 99% were obtained with this device,with sample temperatures staying at or below 106° C. as shown in Table 4below.

TABLE 4 Percent lysis efficiency and sample temperature as function ofpower input. Distance Power (mV) % Lysis In-tube Temp. (° C.) 15 mm 70099.7  78-101 800 99.9 100-101 900 99.8 100-101 1000 99.2 100-102 20 mm700 99.6  95-103 800 99.3  95-104 900 97.7 101-106 1000 97.6 101-105 26mm 700 88.3 45-72 800 97.8 80-92 900 99.7  95-101 1000 99.9  99-101

The sonic field was highly concentrated along the line segment 106 ofthe device 100 except at the ends where the edge effect of thetransducer(s) 104 gave a typical high peak. Measurement of acousticemissions with a pin transducer failed to show characteristic cavitationnoise, supporting a non-cavitation, non-thermal mechanical lysingmechanism. Since there is no correlation between in-tube temperature andlysis efficiency, these results suggest that a continuous-flow,low-temperature, high-efficiency lysis system can indeed be constructedwith judicious selection of transducer(s) 104 and physical geometry.

Experiment 5

An experiment was also conducted to compare lysis efficiency obtainedusing degassed and standard liquid solutions. The device 100 ofexperiment 4 was used and the spore solution was passed through thereaction tube 202 positioned 15 mm above the transducer(s) 104. 200 μlof the spore solution was processed at a flow rate of 1 μl/s. The data,shown in Table 5, shows that greater than 98% lysis efficiency wasachieved for both standard and degassed solutions exposed to twodifferent sonic field intensities. Since the removal of bubbles in thedegassed solution did not significantly decrease the lysis efficiency,this result further supported the hypothesis that cavitation is not theprimary mechanism of spore lysis in the device 100.

TABLE 5 Percent spore lysis efficiency obtained in degassed and standardsolutions. Degassed Power (mV) % Lysis yes 700 98.6 no 700 99.2 yes 80099.3 no 800 99.6

Closure

While embodiments of the present invention have been shown anddescribed, it will be apparent to those skilled in the art that manychanges and modifications may be made without departing from theinvention in its broader aspects. The appended claims are thereforeintended to cover all such changes and modifications as fall within thetrue spirit and scope of the invention.

We claim:
 1. A method for ultrasonically treating a liquid in a flowpath, comprising the steps of: a. positioning at least twoultrasound-producing transducers asymmetrically about a line segment,said at least two transducers producing a concentrated sonic fieldencompassing said line segment; b. providing said flow path for theliquid coincident with said line segment; c. acoustically coupling saidat least two transducers to the liquid in said flow path; d. energizingsaid at least two transducers with a power supply; and e. operating saidat least two transducers at a frequency wherein ultrasonic treatment ofa liquid is from non-thermal and non-cavitating ultrasonic interactions.2. The method of claim 1, wherein said at least two transducersconcentrate sonic energy along said line segment.
 3. The method of claim1, wherein the liquid is flowing continuously in said flow path.
 4. Themethod of claim 1, wherein the liquid is flowing intermittently in saidflow path.
 5. The method of claim 1, wherein the liquid is asingle-phase liquid.
 6. The method of claim 5, wherein said single-phaseliquid is biological.
 7. The method of claim 1, wherein the liquid is aliquid-solid biological material mixture selected from the groupconsisting of suspension, dispersion, slurry, colloid, and biologicaltissue.
 8. The method of claim 1, wherein the liquid is a liquid-solidmixture comprising biological material selected from the groupconsisting of microorganisms, cells, viruses, tissues, and combinationsthereof.
 9. The method of claim 1, wherein the liquid comprises apetrochemical.
 10. The method of claim 1, wherein said treating theliquid is lysing biological material in the liquid.
 11. The method ofclaim 1, wherein said treating the liquid is a chemical process selectedfrom the group consisting of activating, crystallizing, precipitating,and combinations thereof.
 12. The method of claim 1, wherein saidtreating the liquid is a physical process selected from the groupconsisting of sterilizing, extracting, impregnating, dispersing,defoaming, degassing, deaggregating, homogenizing, emulsifying, andcombinations thereof.
 13. The method of claim 1, wherein said at leasttwo transducers operate at a frequency in the range from 0.5 to 5 MHz.14. A method for ultrasonically treating a liquid in a flow path,comprising the steps of: a. positioning at least twoultrasound-producing transducers asymmetrically about a line segment,said at least two transducers producing a concentrated sonic fieldencompassing said line segment; b. providing said flow path for theliquid coincident with said line segment; c. acoustically coupling saidat least two transducers to the liquid in said flow path; d. energizingsaid at least two transducers with a power supply; and e. operating saidat least two transducers at a frequency wherein cavitation cannot bedetected and ultrasonic treatment of a liquid results essentially fromnon-thermal mechanical mechanisms.
 15. A method for ultrasonicallytreating a liquid in a flow path, comprising the steps of: a.positioning at least two ultrasound-producing transducers asymmetricallyabout a line segment, said at least two transducers producing aconcentrated sonic field encompassing said line segment; b. providingsaid flow path for the liquid coincident with said line segment; c.acoustically coupling said at least two transducers to the liquid insaid flow path; d. energizing said at least two transducers with a powersupply;,and e. operating said at least two transducers at a frequencywherein cavitation does not occur and ultrasonic treatment of a liquidresults essentially from non-thermal mechanical mechanisms.